Cognitive decline is emerging as one of the greatest health problems in the elderly population.[1,2] Age alone increases the risk of stroke, Alzheimer's disease (AD), and other forms of dementia [2]. The risk of AD increases 14-fold between the ages of 65-85, and affects almost 47% over the age of 85 [3].
Multiple signaling pathways regulate neuronal survival and growth to facilitate the formation of synapses and this signaling is altered with age [4,5,6,7]. Synapses are essential for learning, memory and the development of neurons in the CNS [8]. Receptors and associated proteins aggregate to mold and shape post-synaptic densities in order to permit high fidelity signal transduction leading to rapid regulation of neuronal function [9,10,11]. Understanding the basic pathophysiological mechanisms of cognitive decline and how the subcellular organization of signaling molecules is altered with cognitive decline could potentially yield novel therapeutic targets for neuronal aging and neurodegeneration.
Cholesterol is a major lipid component of synapses and a limiting factor in synapse development, synaptic activity, and neurotransmitter release [12]. Age-related impairments in the biosynthesis, transport, or uptake of cholesterol by neurons in the CNS may adversely affect development, plasticity, and synaptic circuitry associated with neurodegenerative diseases [13,14,15,16,17]. Membrane lipid rafts (MLR), discrete regions of the plasma membrane enriched in cholesterol, glycosphingolipids and sphingomyelin, are essential for synapse development, stabilization, and maintenance [12,18]. Moreover, caveolin-1 (Cav-1), a cholesterol binding and resident protein of MLR [19,20,21], organizes and targets synaptic components of the neurotransmitter and neurotrophic receptor signaling pathways to MLR [e.g., NMDAR, AMPAR, TrkR, Step Family Kinases (SFK)] [22,23,24,25,26,27]. Additionally, neurotransmitter and neurotrophic receptors are found within MLR in growth cones, a finding that has major implications for neuronal plasticity [11,28].
Early-onset AD, which afflicts individuals prior to 60-65 years of age, is known to be caused by mutations in three genes: amyloid precursor protein (APP), presenilin-1, and presenilin-2 [29]. MLR and cholesterol play a protective role against APP processing and amyloid-β (Aβ) toxicity [13,14,16,30,31,32,33]. Cav-1 KO mice develop CNS pathology similar to AD, such as altered NMDA receptor signaling, motor and behavioral abnormalities, increased ischemic cerebral injury, impaired spatial memory, and cholinergic function [27,34,35,36]. Whether MLR, Cav-1 expression, and the organization of pro-survival and pro-growth signaling mechanisms are altered in neurodegenerative states (age-related dementia and AD) has yet to be investigated. The present study tested whether 1) Cav-1 organizes synaptic signaling components in neuronal MLR and synaptosomes, 2) the localization of synaptic signaling components to neuronal MLR and synaptosomes is reduced in brains from aged wild-type and young Cav-1 KO mice, and 3) brains from Cav-1 KO mice develop a neuropathological phenotype similar to Alzheimer's disease.
The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease. We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression. Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimer's disease.
Decreased expression of pro-survival and growth-stimulatory pathways, in addition to an environment that inhibits neuronal growth, contribute to the limited regenerative capacity in the central nervous system following injury or neurodegeneration. Membrane/lipid rafts, plasmalemmal microdomains enriched in cholesterol, sphingolipids, and the protein caveolin (Cav), are essential for synaptic development/stabilization and neuronal signaling. Cav-1 concentrates glutamate and neurotrophin receptors and pro-survival kinases, and regulates cAMP formation. Here, we show that primary neurons that express a synapsin-driven Cav-1 vector (SynCav1) have increased raft formation, neurotransmitter and neurotrophin receptor expression, NMDA- and BDNF-mediated pro-survival kinase activation, agonist-stimulated cAMP formation, and dendritic growth. Moreover, expression of SynCav1 in Cav-1 KO neurons restores NMDA- and BDNF-mediated signaling and enhances dendritic growth. The enhanced dendritic growth occurred even in the presence of inhibitory cytokines (TNFα, IL-1β) and myelin-associated glycoproteins (MAG, Nogo). Targeting of Cav-1 to neurons thus enhances pro-survival and pro-growth signaling and may be a novel means to repair the injured and neurodegenerative brain.
Multiple signaling pathways have been identified that promote growth and survival of neurons and thereby facilitate the formation of synaptic connections that are essential for learning, memory, and the development of the CNS (Toescu, E. C., Verkhratsky, A., and Landfield, P. W. (2004) Trends Neurosci 27, 614-620; Hattiangady, B., Rao, M. S., Shetty, G. A., and Shetty, A. K. (2005) Exp Neural 195, 353-371; Hotulainen, P., and Hoogenraad, C. C. (2010) J Cell Biol 189, 619-629). Neurotransmitter and neurotrophic receptors, non-receptor tyrosine kinases and other signaling mediators aggregate to mold and shape postsynaptic densities in order to permit high-fidelity signal transduction and the regulation of neuronal function (Huber, A. B., Kolodkin, A. L., Ginty, D. D., and Cloutier, J. F. (2003) Annu Rev Neurosci 26, 509-563; Calabrese, B., Wilson, M. S., and Halpain, S. (2006) Physiology (Bethesda) 21, 38-47; Guirland, C., and Zheng, J. Q. (2007) Adv Exp Med Biol 621, 144-155). A major non-protein component of synapses is cholesterol, which can be a limiting factor in synapse development, synaptic activity, and transmitter release (Mauch, D. H., Nagler, K., Schumacher, S., Goritz, C., Muller, E. C., Otto, A., and Pfrieger, F. W. (2001) Science 294, 1354-1357).
Increasing evidence shows that membrane/lipid rafts, discrete regions of the plasma membrane enriched in cholesterol, glycosphingolipids and sphingomyelin, organize pro-survival and pro-growth neuronal signaling pathways (Allen, J. A., Halverson-Tamboli, R. A., and Rasenick, M. M. (2007) Nat Rev Neurosci 8, 128-140; Head, B. P., Patel, H. H., Tsutsumi, Y. M., Hu, Y., Mejia, T., Mora, R. C., Insel, P. A., Roth, D. M., Drummond, J. C., and Patel, P. M. (2008) Faseb J 22, 828-840; Stern, C. M., and Mermelstein, P. G. (2010) Cell Mol Life Sci 67, 3785-3795), regulate cAMP formation (Oshikawa, J., Toya, Y., Fujita, T., Egawa, M., Kawabe, J., Umemura, S., and Ishikawa, Y. (2003) Am J Physiol Cell Physiol 285, C567-574), and are essential for synapse development, stabilization, and maintenance (Mauch, D. H., Nagler, K., Schumacher, S., Goritz, C., Muller, E. C., Otto, A., and Pfrieger, F. W. (2001) Science 294, 1354-1357; Willmann, R., Pun, S., Stallmach, L., Sadasivam, G., Santos, A. F., Caroni, P., and Fuhrer, C. (2006) Embo J 25, 4050-4060). Caveolin (Cav), a cholesterol binding protein and scaffolding protein found within membrane/lipid rafts (Smart, E. J., Graf, G. A., McNiven, M. A., Sessa, W. C., Engelman, J. A., Scherer, P. E., Okamoto, T., and Lisanti, M. P. (1999) Mol Cell Biol 19, 7289-7304), organizes and targets certain neuronal growth-promoting proteins, such as components of the neurotransmitter and neurotrophic receptor signaling pathways, to membrane/lipid rafts; these include NMDAR, AMPAR, TrkR, GPCRs, Src Family Kinases (SFK)] (Head, B. P., Patel, H. H., Tsutsumi, Y. M., Hu, Y., Mejia, T., Mora, R. C., Insel, P. A., Roth, D. M., Drummond, J. C., and Patel, P. M. (2008) Faseb J 22, 828-840; Bilderback, T. R., Gazula, V. R., Lisanti, M. P., and Dobrowsky, R. T. (1999) J Biol Chem 274, 257-263; Hibbert, Kramer, B. M., Miller, F. D., and Kaplan, D. R. (2006) Mol Cell Neurosci 32, 387-402; Bjork, K., Sjogren, B., and Svenningsson, P. (2010) Exp Cell Res 316, 1351-1356). These receptors and signaling molecules can enhance cAMP formation, an essential second messenger for promoting neuronal growth and dendritic arborization (Neumann, S., Bradke, F., Tessier-Lavigne, M., and Basbaum, A. I. (2002) Neuron 34, 885-893; Wayman, G. A., Impey, S., Marks, D., Saneyoshi, T., Grant, W. F., Derkach, V., and Soderling, T. R. (2006) Neuron 50, 897-909; MacDonald, E., Van der Lee, H., Pocock, D., Cole, C., Thomas, N., VandenBerg, P. M., Bourtchouladze, R., and Kleim, J. A. (2007) Neurorehabil Neural Repair 21, 486-496; Saneyoshi, T., Wayman, G., Fortin, D., Davare, M., Hoshi, N., Nozaki, N., Natsume, T., and Soderling, T. R. (2008) Neuron 57, 94-107; Murray, A. J., Tucker, S. J., and Shewan, D. A. (2009) J Neurosci 29, 15434-15444) and are found within membrane/lipid rafts in growth cones (Guirland, C., and Zheng, J. Q. (2007) Adv Exp Med Biol 621, 144-155). In the setting of traumatic brain injury and neurodegenerative disorders, interventions that activate signaling pathways to stimulate cAMP production thus have the potential to improve functional recovery in such settings (MacDonald, E., Van der Lee, H., Pocock, D., Cole, C., Thomas, N., VandenBerg, P. M., Bourtchouladze, R., and Kleim, J. A. (2007) Neurorehabil Neural Repair 21, 486-496; Atkins, C. M., Oliva, A. A., Jr., Alonso, 0. F., Pearse, D. D., Bramlett, H. M., and Dietrich, W. D. (2007) Exp Neurol 208, 145-158).
A major problem following brain injury (e.g., stroke or trauma) and neurodegeneration is limited functional recovery as a consequence of a reduction in signaling that promotes neuronal growth and survival (Atkins, C. M., Oliva, A. A., Jr., Alonso, O. F., Pearse, D. D., Bramlett, H. M., and Dietrich, W. D. (2007) Exp Neurol 208, 145-158; Hicks, R. R., Zhang, L., Dhillon, H. S., Prasad, M. R., and Seroogy, K. B. (1998) Brain Res Mal Brain Res 59, 264-268; Biegon, A., Fry, P. A., Paden, C. M., Alexandrovich, A., Tsenter, J., and Shohami, E. (2004) Proc Natl Acad Sci USA 101, 5117-5122; Atkins, C. M., Falo, M. C., Alonso, O. F., Bramlett, H. M., and Dietrich, W. D. (2009) Neurosci Lett 459, 52-56). This loss of “protective signaling” increases neuronal loss, impairs brain repair, and increases functional deficits. Therapeutic interventions, such as addition of growth factors or approaches to increase cAMP, are relatively ineffective because of the loss of key receptors and their downstream signaling molecules. Therefore, interventions that restore pro-growth and pro-survival signaling within neurons have the potential not only to reduce neuronal loss and enhance endogenous brain repair, but also to increase the efficacy of pharmacologic agents designed to improve functional outcome (Carmichael, S. T. (2008) Stroke 39, 1380-1388).
We show that over-expression of neuron-targeted Cav-1 in primary neurons enhances expression of membrane/lipid rafts, neurotransmitter and neurotrophin receptors and increases pro-growth signaling, cAMP production, and dendritic growth and arborization. Conversely, siRNA-mediated loss of Cav-1 decreases membrane/lipid rafts and expression of neurotransmitter and neurotrophin receptors, and blunts NMDA- and BDNF-mediated signaling and attenuates agonist-stimulated cAMP production. Re-expression of Cav-1 in Cav-1 KO primary neurons restores pro-survival signaling and promotes neuronal growth and arborization even in the presence of inhibitory cytokines and myelin-associated glycoproteins. These growth-promoting effects of neuron-targeted Cav-1 expression suggest that it might be useful as a therapeutic intervention to limit neurodegeneration and to enhance repair of the injured CNS.